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Title: Accumulation and degradation of aliphatic nitrocompounds in Hippocrepis comosa and other legumes
Author: Salem, M. A.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 1997
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3-nitropropionic acid was surveyed in leaves of 155 angiosperm species representing 50 families (mostly European). 3-nitropropionate was found only in extracts of 9 legumes. One of these was Hippocrepis comosa which was studied in detail. 3-nitropropionic acid and three of its glucose esters 6-O-(3-nitropropanoyl)-αβ-D-glucopyranose; 1,6-di-O-(3-nitropropanoyl)-β-D-glucopyranose (cibarian) 1,2,6-tri-O-(3-nitropropanoyl)-β-D-glucopyranose (karakin) were isolated from shoots of this species and identified spectrophotometrically. In addition, the 3-nitropropanoyl ester of 4-hydroxybutanoic acid was extracted and identified. This was the first isolation of this compound from a natural source. 3NPA was found in different concentration in leaves, roots and other parts of H. camosa. In experiments where H. comosa was grown in the growth room in water culture containing ammonium, urea or nitrate as nitrogen nutrient, it was shown that the source of nitrogen for growth did not affect the accumulation of 3NPA to any significant extent. An enzyme activity that catalyses the oxidation of 3NPA was isolated and characterised. This enzyme, named 3-nitropropionic acid oxidase here, oxidised 3NPA to nitrite and nitrate. Hydrogen peroxide and malonate semialdehyde were also products of the reaction. The enzyme 3NPA oxidase was purified to electrophoretic homogeneity from leaves of H. comosa after a 5 step procedure involving ammonium sulphate fractionation, dialysis, mono Q-sepharose anion exchange chromatography (FPLC), ultrafiltration and superose 12 gel filtration (FPLC). The subunit of the enzyme has a molecular weight of 36.3 kDa and the enzyme has an apparent Km for 3NPA of 688 μM. Of 10 aliphatic nitrocompounds tested only 3NPA acted as substrate for the enzyme. Stoichiometry studies showed that 3 moles of 3NPA is converted to 1.0 moles of nitrite, 2.0 moles of nitrate, 0.5 moles of hydrogen peroxide and 3.0 moles of malonate semi aldehyde with consumption of 3.0 moles of oxygen.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available